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RESEARCHNOTE BACTERIOLOGY
Evaluation of different culture methods for of fungal peritonitis [1,2]. Culture of CAPD fluid helps in
the diagnosis of peritonitis in patients on selecting an antimicrobial agent and to identify the source of
continuous ambulatory peritoneal dialysis infection [1]. Various culture methods have been employed to
increase the sensitivity of culture by inoculating the centrifuged
deposit of fluid in different culture media and inoculating the
1 2 3 3 fluid in blood culture bottles [3–5].
R. N. Iyer , A. K. Reddy , S. Gande and A. Aiyangar
1) Department of Microbiology, Global Hospitals, 2) Department of Among the different culture methods, inoculation of blood
Microbiology, GHR Micro Diagnostics and 3) Department of Nephrology, culture bottles with the fluid was observed in the past to
Global Hospitals, Hyderabad, India enhance the yield of organisms [3–5]. The presence of
organisms sequestered within phagocytes such as polymorphs
precludes the isolation of organisms and could be a reason for
false-negative results, despite the use of automated blood
culture systems [6]. Water lysis, incorporation of Tween 80 in
Abstract blood agar and treatment of specimens with Triton-X anionic
surface active agents have been described as options for
A total of 170 continuous ambulatory peritoneal dialysis (CAPD) increasing the yield of clinically significant organisms, by
fluids were processed by various culture methods, including direct disrupting phagocytes and subsequent release of intracellular
inoculation of the centrifuged sediment, inoculation into auto- organisms [6]. However, all methods have been tried in
mated blood culture bottles, water lysis, Tween-80 incorporated isolation and a combination of methods has not been
blood agar, and Triton-X treatment of the specimen. Of 170 attempted. The aim of this study was to evaluate the
CAPDfluids,127showedthegrowthofbacteria/fungi. Sixty-three performance of different culture methods for the diagnosis
fluids showed growth by all methods, the water lysis alone of peritonitis in patients on CAPD.
detected 24 additional positive cultures, while Tween-80 blood A total of 170 CAPD effluent bags received between
agar and Triton-X yielded 30 additional positive cultures. A January 2009 and September 2010 were included in the study.
combination of water lysis, Tween-80 blood agar and Triton-X All effluent bags were transported to the Department of
treatment of the CAPD fluid is recommended for diagnosis of Microbiology within 30 min of dialysis and all bags were
CAPD peritonitis in resource-limited settings. inspected for obvious leaks on receipt at the laboratory.
Around 100 mL of fluid was aspirated from the injection port
Keywords: BACTEC, CAPD, Triton-X, Tween-80, water lysis of the CAPD bag under all aseptic precautions. Ten mL of the
Original Submission: 14 June 2013; Revised Submission: aspirated fluid was inoculated into the BACTEC Aerobic F
22 August 2013; Accepted: 16 September 2013 bottles and the BACTEC Mycoses bottles and incubated in the
Editor: D. Raoult BACTEC 9120 (BD & Co, Sparks, MD, USA) for 7 days. The
Article published online: 11 November 2013 remaining fluid was distributed aseptically into 15-mL sterile
Clin Microbiol Infect 2014; 20: O294–O296 centrifuge tubes and was processed as per the following
10.1111/1469-0691.12402 protocol. Tubes were centrifuged at 134.1 g for 10 min, the
supernatant was discarded and the sediments were pooled and
recentrifuged at the same speed for 10 min before final
Correspondingauthor:DrRanganathanNIyer,ConsultantClinical sediment was obtained. Loopfuls of the sediment were
Microbiologist, Global Hospitals, Lakdi- ka- pool, Hyderabad 50004, inoculated into sheep blood agar (5% sheep blood), chocolate
India
E-mail: ranganathaniyer@yahoo.com agar, Tween-80 incorporated blood agar, brain heart infusion
agar and Sabourauds dextrose agar.
The procedure for water lysis includes 50 mL of the fluid in
Peritonitis is a common complication in patients with end- two centrifuge tubes, which were centrifuged at 134.1 g for
stage renal disease on continuous ambulatory peritoneal 10 min and the supernatant discarded. The sediment was
dialysis (CAPD), contributing to 16% of deaths in these resuspended in 10 mL of sterile distilled water with vigorous
patients [1]. Episodes of peritonitis are best managed with the shaking for 15 min. This was recentrifuged and the deposit was
help of good clinical microbiology laboratory support with suspended. This was inoculated into blood and chocolate agar
direct microscopy and culture of CAPD fluids. Whilst direct [7]. Fifteen mL of fluid in a centrifuge tube was used for the
microscopy has a limited role in the diagnosis of bacterial Triton-X treatment [6]. Two or three drops of Triton-X
peritonitis, it is useful for a prompt diagnosis and management (Sigma reagent grade) was added to 15 mL fluid and allowed to
ª2013 The Authors
Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases
CMI Research Note O295
stand with intermittent shaking at 37°C for 15 min. This was TABLE 2. Organisms isolated from the CAPD cultures
inoculated into blood and chocolate agar. Blood agar (n = 127)
containing Tween-80 was prepared by adding 2% Tween-80 Organisms isolated No. (%)
to the Columbia blood agar base and the mixture was
autoclaved at 121°C for 15 min, at a pressure of 15 pounds Staphylococcus aureus 34 (26.7)
Enterococcus faecalis 23 (18.1)
per square inch. Sheep blood (5%) was added after the basal E. coli 19 (14.9)
Klebsiella pneumoniae 12 (9.4)
medium cooled to 45°C and the medium poured. Loopfuls of Enterobacter cloacae 7 (5.5)
Citrobacter freundii 1 (0.78)
the centrifuged deposit were inoculated and all plates were Pseudomonas aeruginosa 8 (6.29)
Acinetobacter baumanii calcoaceticus complex 7 (5.5)
incubated aerobically at 37°C with added 5–6% CO2 for 48 h Serratia marcescens 1 (0.78)
Candida albicans 4 (3.14)
[8]. The inoculated fungal culture media were incubated at 35° Candida parapsilosis 6 (4.7)
Cfor 24 h and then at 28–30°C for another 3 weeks before Candida tropicalis 2 (1.57)
being discarded. All plates were inspected at 24-h intervals for
growth of organisms. The remaining sediment after the direct
centrifugation of the fluid was used for the direct microscopic The low culture positivity in patients with peritonitis on
examination, which included gram stain and other special CAPDhasbeenattributed to the low load of the organisms in
stains. The statistical significance was calculated. the majority of the patients, the presence of an antimicrobial
Of 170 CAPD fluids, 127 showed the growth of bacteria, agent in the peritoneal fluid of the patient receiving antimi-
which were identified using standard biochemical reactions, crobial therapy, the presence of intracellular organisms,
and yeast isolates, using a combination of germ tube, assim- peritonitis caused by endotoxin released by bacterial infection
ilation reactions and morphology on corn meal agar. The and the culture technique used for the isolation of the
culture positivity identified using different culture methods is organism [9]. Different investigators have used several meth-
shown in Table 1. Of 127 culture-positive fluids, 63 showed ods to enhance the yield of organisms from the CAPD effluent,
growth using the conventional centrifugation culture method including the use of mass spectroscopy and sequencing and
and all these fluids also showed growth when using the MALDI-TOF [3–11]. In the present study, we have observed
remaining four methods. Inoculation of fluids into BACTEC high culture positivity with water lysis specimens and speci-
bottles detected growth in an additional 10 patients compared mens pretreated with Triton-X and Tween-80 incorporated
with the conventional culture method. Incorporation of blood agar when compared with the automated blood culture
Tween-80 and Triton-X treatment resulted in culture positiv- system and direct inoculation of the centrifuged deposit of the
ity in 30 additional fluids. There was no difference in the specimen into different culture media. This was also shown to
culture positivity rate between the Triton-X and Tween 80 be statistically significant. The high culture positivity of the
methods. The water lysis method detected 24 additional above methods is due to the release of intracellular organisms
positive cultures and all these showed no growth by Triton-X/ present in phagocytes by prior treatment of the specimen with
Tween-80. The 30 fluids that showed growth using the chemical and physical methods. However, there are concerns
Tween-80 and Triton-X methods showed no growth when that manipulation of specimens prior to inoculation may result
using the water lysis method. The fluids that showed growth in contamination and this can be overcome by following
using the BACTEC method also showed growth when using standard microbiology precautions.
the Triton X/Tween 80 incorporated blood agar and water Several studies have shown that culture rates increased
lysis method. The organisms isolated are shown in Table 2. significantly with the BACTEC blood culture system compared
with conventional culture systems [3,5,9,12]. In our study, we
also found that the BACTEC blood culture system increased
TABLE 1. Culture positivity by different culture methods the culture positivity (73/127) compared with direct inocula-
(n = 127) tion of the centrifuged deposit of fluid into different culture
No. of positive cultures (%), media (63/127). Although the BACTEC method allows the
Culture method p value
rapid detection of pathogens, it requires expensive equipment
Direct culture from the sediment 63 (49.6) and blood culture bottles. A cost analysis of the different
BACTEC blood culture bottle inoculation 73 (57.4)
Tween 80 blood agar 93 (73.2), <0.0001 methods revealed the water analysis and the use of Tween 80
Triton X-treated specimens 93 (73.2), <0.0001
Water lysis 87 (68.5), <0.001 blood agar and Triton X (at Rs 250/- and Rs 50/-, respectively)
Combination of Triton X/Tween 80 and 117 (92.1), <0.0001
water lysis to be less expensive compared with inoculating blood culture
bottles (Rs 500/-).
ª2013 The Authors
Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20, O294–O296
O296 Clinical Microbiology and Infection, Volume 20 Number 5, May 2014 CMI
We have retrospectively reviewed the white blood cell 2. Predari SC, De Paulis AN, Veron D, Zucchini A, Santoianni JE. Fungal
(WBC)countsofthe 170 fluids received for culture and found peritonitis in patients on peritoneal dialysis: twenty five years of
–1 experience a teaching hospital in Argentina. Rev Argent Microbiol 2007;
that WBC count was less than 100 lL and polymorphonu- 39: 213–217.
clear leukocyte counts were less than 50% in 30/43 culture- 3. Yoon SH, Choi NW, Yun SR. Detecting bacterial growth in continuous
negative patients. This explains the low culture positivity (127/ ambulatory peritoneal dialysis effluent using two culture methods.
70) of CAPD fluids in our study compared with other studies Korean J Intern Med 2010; 25: 82–85.
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our results, we recommend the use of inoculation of the 1993; 83: 42–43.
centrifuged deposit into different culture media and pretreat- 5. Males BM, Walshe JJ, Garringer L, Koscinski D, Amsterdam D. Addi-
Chek filtration, BACTEC, and 10-ml culture methods for recovery of
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continuous ambulatory peritoneal dialysis peritonitis effluent after
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ª2013 The Authors
Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20, O294–O296
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